Secondary structure stabilized NMDA receptor modulators and uses thereof

ABSTRACT

Disclosed are compounds having enhanced potency in the modulation of NMDA receptor activity. Such compounds are contemplated for use in the treatment of diseases and disorders such as learning, cognitive activities, and analgesia, particularly in alleviating and/or reducing neuropathic pain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofPCT/US2011/24583, filed Feb. 11, 2011, which claims priority to U.S.Ser. No. 61/303,472, filed Feb. 11, 2010, all of which are incorporatedby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 10, 2014, isnamed NAU-004_SL.txt and is 3,895 bytes in size.

BACKGROUND

An N-methyl-d-aspartate (NMDA) receptor is a postsynaptic, ionotropicreceptor that is responsive to, inter alia, the excitatory amino acidsglutamate and glycine and the synthetic compound NMDA. The NMDA receptorcontrols the flow of both divalent and monovalent ions into thepostsynaptic neural cell through a receptor associated channel (Fosteret al., Nature 1987, 329:395-396; Mayer et al., Trends in Pharmacol.Sci. 1990, 11:254-260). The NMDA receptor has been implicated duringdevelopment in specifying neuronal architecture and synapticconnectivity, and may be involved in experience-dependent synapticmodifications. In addition, NMDA receptors are also thought to beinvolved in long term potentiation and central nervous system disorders.

The NMDA receptor plays a major role in the synaptic plasticity thatunderlies many higher cognitive functions, such as memory acquisition,retention and learning, as well as in certain cognitive pathways and inthe perception of pain (Collingridge et al., The NMDA Receptor, OxfordUniversity Press, 1994). In addition, certain properties of NMDAreceptors suggest that they may be involved in theinformation-processing in the brain that underlies consciousness itself.

The NMDA receptor has drawn particular interest since it appears to beinvolved in a broad spectrum of CNS disorders. For instance, duringbrain ischemia caused by stroke or traumatic injury, excessive amountsof the excitatory amino acid glutamate are released from damaged oroxygen deprived neurons. This excess glutamate binds to the NMDAreceptors which opens their ligand-gated ion channels; in turn thecalcium influx produces a high level of intracellular calcium whichactivates a biochemical cascade resulting in protein degradation andcell death. This phenomenon, known as excitotoxicity, is also thought tobe responsible for cardiac arrest to epilepsy. In addition, there arepreliminary reports indicating similar involvement in the chronicneurodegeneration of Huntington's, Parkinson's, and Alzheimer'sdiseases. Activation of the NMDA receptor has been shown to beresponsible for post-stroke convulsions, and, in certain models ofepilepsy, activation of the NMDA receptor has been shown to be necessaryfor the generation of seizures. Neuropsychiatric involvement of the NMDAreceptor has also been recognized since blockage of the NMDA receptorCa⁺⁺ channel by the animal anesthetic PCP (phencyclidine) produces apsychotic state in humans similar to schizophrenia (reviewed in Johnson,K. and Jones, S., 1990). Further, NMDA receptors have also beenimplicated in certain types of spatial learning.

The NMDA receptor is believed to consist of several protein chainsembedded in the postsynaptic membrane. The first two types of subunitsdiscovered so far form a large extracellular region, which probablycontains most of the allosteric binding sites, several transmembraneregions looped and folded so as to form a pore or channel, which ispermeable to Ca⁺⁺, and a carboxyl terminal region. The opening andclosing of the channel is regulated by the binding of various ligands todomains (allosteric sites) of the protein residing on the extracellularsurface. The binding of the ligands is thought to affect aconformational change in the overall structure of the protein which isultimately reflected in the channel opening, partially opening,partially closing, or closing.

NMDA receptor compounds may exert dual (agonist/antagonist) effect onthe NMDA receptor through the allosteric sites. These compounds aretypically termed “partial agonists”. In the presence of the principalsite ligand, a partial agonist will displace some of the ligand and thusdecrease Ca⁺⁺ flow through the receptor. In the absence of or loweredlevel of the principal site ligand, the partial agonist acts to increaseCa⁺⁺ flow through the receptor channel.

A need continues to exist in the art for novel and more specific/potentcompounds that are capable of binding to, or modulating the glycinebinding site of NMDA receptors, e.g the NMDA receptor NR1 ligand bindingcore, with e.g. with significant specificity and/or potency, especiallyin-vivo, to provide pharmaceutical benefits. In addition, a needcontinues to exist in the medical arts for an orally deliverable formsof such compounds.

SUMMARY

Provided herein, at least in part, are compounds that are NMDAmodulators, for example, partial agonists of NMDA. For example, providedherein are compounds that can mimic a beta-turn structure that iscapable of selectively interacting with the glycine binding region ofNMDA receptor NR1, e.g. SEQ ID. NO. 1. Disclosed peptide mimetics, forexample, have a beta-turn motif when binding to SEQ ID NO. 1. In someembodiments, disclosed peptide mimetics substantially maintain abeta-turn motif in vivo or in an aqueous solution.

In some embodiments, a peptide mimetic capable of binding to, orassociating with, the NMDA ligand binding core of SEQ ID NO. 1 isprovided, wherein said peptide mimetic has at least two alpha carbonsabout 6 to about 14 Å apart, e.g., about 6 to about 8 Å apart. Forexample, disclosed mimetics may include a cyclic amide core, e.g. aspiro-beta-lactam.

In another embodiment, a disclosed peptide mimetic may be a peptidehaving two or three amino acids replaced with a moiety having a carboxylgroup and an amino group. In an embodiment, a disclosed peptide mimeticof any one of claims 1-6, where said peptide mimetic is capable offorming a hydrogen bond at least one, two, three or four of thefollowing amino acids of SEQ ID NO.1: PRO124, THR126, GLU178 and SER180,or may be capable of forming a hydrogen bond with all four amino acids.

For example, provided herein is a peptide mimetic capable of binding tothe NMDA ligand binding core of SEQ ID NO. 1, wherein said peptidemimetic has two alpha carbons about 6 to about 14 Å apart, (e.g., about6 to about 10 Å apart) and a beta-turn motif comprising a bicyclic amidecore (e.g., a spiro-beta-lactam) such that, when the peptide mimeticbinds to said SEQ ID NO. 1, the bicyclic amide core substantiallyretains configuration. Such peptide mimetic may include a corerepresented by:

Exemplary peptide mimetics may substantially maintains the beta-turnmotif in vivo or in an aqueous solution, and may be capable of forming ahydrogen bond with the following amino acids of SEQ ID NO.1: PRO124,THR126, GLU178 and SER180. In some embodiments, the beta-turn core maybe conjugated to one or two amino acids.

Methods of treating or preventing a NMDA receptor mediated disorders ina patient are also provided, comprising administering to a patient inneed thereof an acceptable NMDA ligand binding core receptor agonisticor antagonistic amount of a glycine mimicking beta-turn peptidomimeticcyclic compound having a cyclic amide moiety, for example, a beta-lactammoiety.

Also provided herein is a method of modulating the activity of SEQ IDNO. 1, wherein the modulation arises from a favorable conformationadopted by a compound, and wherein said modulation arises from ahydrogen bonding interaction between the compound and one, two, three orfour of the following amino acids of SEQ ID NO.1: PRO124, THR126, GLU178and SER180.

In another embodiment, a method of identifying a compound capable ofbinding to SEQ ID NO. 1 is provided, comprising: a) providing amolecular model comprising one or more target regions of SEQ ID NO. 1derived from at least a portion of: SEQ ID NO. 1, atomic coordinates bymolecular modeling of SEQ ID NO. 1, or atomic coordinates deposited inthe Protein Data Bank under accession number 1PBQ; b) using themolecular model to identify a compound that can bind to the one or moretarget regions in the molecular model; and c) producing the compound. Insome embodiments, such a method may further comprise the additional stepof determining whether the compound modulates SEQ ID NO. 1.

Also provided herein are pharmaceutically acceptable compositionscomprising a disclosed compound, and a pharmaceutically acceptableexcipient. For example, such compositions may be suitable for oraladministration to a patient.

A method for treating a cognitive disorder, such as a disorderassociated with memory loss or impaired learning comprisingadministering to an patient in need thereof an effective amount of adisclosed compound. For example, provided herein are methods of treatingor ameliorating memory loss or impaired learning in a patient in needthereof.

In an embodiment, methods for treating neuropathic pain in a patient inneed thereof comprising administering an effective amount of a disclosedcompound is provided.

Also disclosed herein are methods for treating depression,obsessive-compulsive disorder, or schizophrenia in a patient in needthereof comprising administering an effective amount of a disclosedcompound. In another embodiment, methods for treating post traumaticstress disorder, an alcohol dependency disorder, or an addiction to anaddictive drug in a patient in need thereof comprising administering aneffective amount of a disclosed compounds are provided.

DESCRIPTION OF FIGURES

FIGS. 1A-1D indicate that a disclosed compound (AK52) biphasicallyalters postsynaptic NMDA receptor-mediated excitatory postsynapticcurrents (e.p.s.c.s) at Shaffer collateral-CA1 synapses, and selectivelyenhances induction of LTP. 1A: Time course of the marked reduction byAK52 (1 μM; solid bar) of the NMDA component of Schaffercollateral-evoked e.p.s.c.s in CA1 pyramidal neurons. (Each point is themean±SEM of e.p.s.c. peNRXe amplitude of 5 cells.) 1B: Time course ofthe enhancement of a ten-fold lower concentration of AK52 (100 NM; greybar) of the NMDA component of Schaffer collateral-evoked e.p.s.c.s. inCA1 pyramidal neurons. (Each point is the mean±SEM of e.p.s.c. peNRXamplitude of 5 cells). 1C: Time course of LTD induced by a low frequencystimulus train (2 Hz/10 min; Starting at arrow) at Schaffercollateral-CA1 synapses in slices pre-treated with 1 μM (filled circles;n=10) and 100 nM (filled diamonds; n=6) NRX-10,052, compared to control,untreated slices (open circles; n-8). (Each point is the mean±SEM ofnormalized extracellular field EPSP slope of n slices.) 1D: Time courseof experiments comparing LTP induced by a high frequency stimulus train(3×100 Hz/500 ms; arrow) at Schaffer collateral-CA1 synapses in slicespre-treated with 1 μM (filled circles; n=10 or 100 nM (filled diamonds;n=8) NRX-10,052, compared to control, untreated slices (open circles;n=15). (Each point is the mean±SEM of normalized field e.p.s.p. lsope ofn slices).

FIGS. 2A-2E indicate a low concentration of a disclosed compound Bmarkedly enhances pharmacologically-isolated postsynaptic NMDAreceptor-mediated excitatory postsynaptic currents (e.p.s.c.s) atShaffer collateral-CA1 synapses and potentiates LTP, while a 20-foldhigher concentration reduces NMDA e.p.s.c.s. 2A: Time course of themarked enhancement by Compound B (50 nM; solid bar) of single shockSchaffer collateral-evoked pharmacologically-isolated NMDA e.p.s.c.s.recorded in CA1 pyramidal neurons. 2B: Time course of the enhancement bycompound B (50 nM; solid bar) of burst-evoked (4 pulses/100 Hz) NMDAe.p.s.c.s. 2C: Time course of the marked reduction by compound B (1 μM;solid bar) of single shock Schaffer collateral-evoked NMDA e.p.s.c.s.recorded in CA1 pyramidal neurons. 2D: Time course of the reduction bycompound B (1 μM; solid bar) of burst-evoked (4 pulses 100 Hz) Schaffercollateral-evoked NMDA e.p.s.c.s recorded in CA1 pyramidal neurons. 2E:Enhancement of high frequency (100 Hz/500 ms×3; solid arrow) Schaffercollateral stimulus-evoked LTP at synapses on CA1 pyramidal neurons by50 nM Compound B (filled circles) compared to control, untreated slices(open circles). (Each point is the mean±SEM of e.p.s.c. peNRX amplitudeof n cells.).

FIGS. 3A-3C demonstrate 100 nM and 1 μM concentrations of a disclosedcompound (AK51) both enhance pharmacologically-isolated postsynapticNMDA receptor-mediated (e.p.s.c.s.) at Shaffer collateral-CA1 synapseand potentiate LTP. 3A: Time course of the marked enhancement byNRX-10,051 (100 nM; solid bar) of single shock Schaffercollateral-evoked pharmacologically-isolated NMDA e.p.s.c.s recorded inCA1 pyramidal neurons (n=x). 3B: Time course of the enhancement by AK51(1 μM; solid bar) of single shock Schaffer collateral-evokedpharmacologically-isolated NMDA e.p.s.c.s recorded in CA1 pyramidalneurons (n=y). 3C: Enhancement of high frequency (100 Hz/500 ms×3; solidarrow) Schaffer collateral stimulus-evoked LTP at synapses on CA1pyramidal neurons by 100 nM ( ) and 1 μM (filled circles) AK5151,compared to control, untreated slices (open circles). 3D: Time course ofLTD induced by a low frequency stimulus train (2 Hz/10 min; starting atarrow) at Schaffer collateral-CA1 synapses in slices pre-treated with 1μM (filled circles; n=10) or 100 nM (filled diamonds; n=6 NRX-10,051,compared to control, untreated slices (open circles; n=8). Each point isthe mean±EM of e.p.s.c. peNRX amplitude of n cells.).

FIG. 4 indicates that a disclosed compound enhances NMDA current andLTP. A: Time course of effect of 20 min bath application of 100 nM AK51(solid bar) on normalized pharmacologically-isolated NMDA receptor-gatedcurrent in CA1 pyramidal neurons under whole-cell recording (mean±SEM,n=5). B: Time course of effect of 20 min bath application of 1 μM AK51(solid bar) on normalized pharmacologically-isolated NMDA receptor-gatedcurrent in CA1 pyramidal neurons under whole-cell recording (mean±SEM,n=6). C: Time course of effect of bath application of 100 nM AK51 (solidbar, filled circles, n=8) compared to untreated control slices (opencircles, n=6) on the magnitude of long-term potentiation (LTP) ofextracellular excitatory postsynaptic potential slope (mean±SEM, fEPSP)induced by high-frequency Schaffer collateral stimulation (arrow, 2×100Hz/500 msec). D: Time course of effect of bath application of 1 μM AK51(solid bar, filled circles, n=8) compared to untreated control slices(open circles, n=6) on the magnitude of LTP of fEPSP slope (mean±SEM)induced by high-frequency Schaffer collateral stimulation (arrow, 2×100Hz/500 msec). E: Time course of effect of bath application of 1 μM AK51(solid bar, filled circles, n=10) compared to untreated control slices(open circles, n=8) on the magnitude of long-term depression of fEPSPslope (mean±SEM) induced by low-frequency Schaffer collateralstimulation (arrow, 2 Hz/10 min)

FIG. 5 depicts the results of a T-maze test in rats using a disclosedcompound.

FIG. 6 depicts the results of a formalin neuropathic pain assay in rats.

FIG. 7 indicates that one isomer of a disclosed compound AK-55-Apotently enhances NMDA current and LTP, while AK-55-B does not.

FIG. 8 depicts quantification by GC/MS and shows the area under thecurve for AK-51 and [2H7]proline internal standard and was analyzed withGC/MS by selective ion monitoring following TBDMS derivatization basedon methods adapted from Wood et al. Journal of Chromatography B, 831,313-9 (2005). The quantitative range of the assay for this compound was0.312 pmol to 10 pmol column. The ions utilized for SIM were 241.2 (thiscompound) and 350.3 (deuterated proline). R2=0.9998 (Quadratic non-linerregression).

FIG. 9 depicts the sequence of the NMDA receptor NR1 and variouscompounds that associate via hydrogen bonding to specific amino acids.

FIG. 10 depicts the crystal structure of compound X (GLYX-13) with theNMDA receptor NR1.

FIG. 11 depicts a model peptide (GLYX-13) that has a distance of 12.171Å between alpha carbons.

FIG. 12 depicts a ¹H NMR spectra of a compound disclosed herein.

FIG. 13 depicts a ¹H NMR spectra of a compound disclosed herein.

DETAILED DESCRIPTION

This disclosure is generally directed to compounds that are capable ofmodulating NMDA, e.g. NMDA antagonists or partial agonists, andcompositions and/or methods of using the disclosed compounds.

The following definitions are used throughout the description of thepresent disclosure:

The term “alkenyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon double bond, suchas a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms,referred to herein as C₂-C₁₂alkenyl, C₂-C₁₀alkenyl, and C₂-C₆alkenyl,respectively. Exemplary alkenyl groups include, but are not limited to,vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl,hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl,4-(2-methyl-3-butene)-pentenyl, etc.

The term “alkoxy” as used herein refers to an alkyl group attached to anoxygen (—O-alkyl). Exemplary alkoxy groups include, but are not limitedto, groups with an alkyl group of 1-12, 1-8, or 1-6 carbon atoms,referred to herein as C₁-C₁₂alkoxy, C₁-C₈alkoxy, and C₁-C₆alkoxy,respectively. Exemplary alkoxy groups include, but are not limited tomethoxy, ethoxy, etc. Similarly, exemplary “alkenoxy” groups include,but are not limited to vinyloxy, allyloxy, butenoxy, etc.

The term “alkyl” as used herein refers to a saturated straight orbranched hydrocarbon. Exemplary alkyl groups include, but are notlimited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl,2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,hexyl, heptyl, octyl, etc.

Alkyl, alkenyl and alkynyl groups can optionally be substituted, if notindicated otherwise, with one or more groups selected from alkoxy,alkyl, cycloalkyl, amino, halogen, and —C(O)alkyl. In certainembodiments, the alkyl, alkenyl and alkynyl groups are not substituted,i.e., they are unsubstituted.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond.Exemplary alkynyl groups include, but are not limited to, ethynyl,propynyl, and butynyl.

The term “amide” or “amido” as used herein refers to a radical of theform —R_(a)C(O)N(R_(b))—, —R_(a)C(O)N(R_(b))R_(c)—, or —C(O)NR_(b)R_(c),wherein R_(a), R_(b) and R_(c) are each independently selected fromalkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl,heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, and nitro. Theamide can be attached to another group through the carbon, the nitrogen,R_(b), R_(c), or R_(a). The amide also may be cyclic, for example R_(b)and R_(c), R_(a) and R_(b), or R_(a) and R_(c) may be joined to form a3- to 12-membered ring, such as a 3- to 10-membered ring or a 5- to6-membered ring. The term “carboxamido” refers to the structure—C(O)NR_(b)R_(c).

The term “amine” or “amino” as used herein refers to a radical of theform —NR_(d)R_(e), where R_(d) and R_(e) are independently selected fromhydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl,haloalkyl, heteroaryl, and heterocyclyl. The amino also may be cyclic,for example, R_(d) and R_(e) are joined together with the N to form a 3-to 12-membered ring, e.g., morpholino or piperidinyl. The term aminoalso includes the corresponding quaternary ammonium salt of any aminogroup, e.g., —[N(Rd)(Re)(Rf)]+. Exemplary amino groups includeaminoalkyl groups, wherein at least one of R_(d), R_(e), or R_(f) is analkyl group. In certain embodiment, R_(d) and R_(e) are hydrogen oralkyl.

The terms “halo” or “halogen” or “Hal” as used herein refer to F, Cl,Br, or I. The term “haloalkyl” as used herein refers to an alkyl groupsubstituted with one or more halogen atoms.

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to saturated or partially unsaturated 3- to 10-membered ringstructures, alternatively 3- to 7-membered rings, whose ring structuresinclude one to four heteroatoms, such as nitrogen, oxygen, and sulfur.Heterocycles may also be mono-, bi-, or other multi-cyclic ring systems.A heterocycle may be fused to one or more aryl, partially unsaturated,or saturated rings. Heterocyclyl groups include, for example, biotinyl,chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl,dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl,isothiazolidinyl, isoxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl,phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl,pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl,pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl,tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl,thiopyranyl, xanthenyl, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringmay be substituted at one or more positions with substituents such asalkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl,arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl,ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl,hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato,sulfate, sulfide, sulfonamido, sulfonyl and thiocarbonyl. In certainembodiments, the heterocyclic group is not substituted, i.e., theheterocyclic group is unsubstituted.

The term “heterocycloalkyl” is art-recognized and refers to a saturatedheterocyclyl group as defined above. The term “heterocyclylalkoxy” asused herein refers to a heterocyclyl attached to an alkoxy group. Theterm “heterocyclyloxyalkyl” refers to a heterocyclyl attached to anoxygen (—O—), which is attached to an alkyl group.

The terms “hydroxy” and “hydroxyl” as used herein refers to the radical—OH.

“Pharmaceutically or pharmacologically acceptable” include molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. “For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards

As used in the present disclosure, the term “partial NMDA receptoragonist” is defined as a compound that is capable of binding to aglycine binding site of an NMDA receptor; at low concentrations a NMDAreceptor agonist acts substantially as agonist and at highconcentrations it acts substantially as an antagonist. Theseconcentrations are experimentally determined for each and every “partialagonist.

As used herein “pharmaceutically acceptable carrier” or “exipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. In one embodiment, the carrieris suitable for parenteral administration. Alternatively, the carriercan be suitable for intravenous, intraperitoneal, intramuscular,sublingual or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The term “pharmaceutically acceptable salt(s)” as used herein refers tosalts of acidic or basic groups that may be present in compounds used inthe present compositions. Compounds included in the present compositionsthat are basic in nature are capable of forming a wide variety of saltswith various inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.Compounds included in the present compositions that include an aminomoiety may form pharmaceutically acceptable salts with various aminoacids, in addition to the acids mentioned above. Compounds included inthe present compositions that are acidic in nature are capable offorming base salts with various pharmacologically acceptable cations.Examples of such salts include alkali metal or alkaline earth metalsalts and, particularly, calcium, magnesium, sodium, lithium, zinc,potassium, and iron salts.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S,” depending on the configuration of substituentsaround the stereogenic carbon atom. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.Stereoisomers include enantiomers and diastereomers. Mixtures ofenantiomers or diastereomers may be designated “(±)” in nomenclature,but the skilled artisan will recognize that a structure may denote achiral center implicitly.

Individual stereoisomers of compounds of the present invention can beprepared synthetically from commercially available starting materialsthat contain asymmetric or stereogenic centers, or by preparation ofracemic mixtures followed by resolution methods well known to those ofordinary skill in the art. These methods of resolution are exemplifiedby (1) attachment of a mixture of enantiomers to a chiral auxiliary,separation of the resulting mixture of diastereomers byrecrystallization or chromatography and liberation of the optically pureproduct from the auxiliary, (2) salt formation employing an opticallyactive resolving agent, or (3) direct separation of the mixture ofoptical enantiomers on chiral chromatographic columns. Stereoisomericmixtures can also be resolved into their component stereoisomers by wellknown methods, such as chiral-phase gas chromatography, chiral-phasehigh performance liquid chromatography, crystallizing the compound as achiral salt complex, or crystallizing the compound in a chiral solvent.Stereoisomers can also be obtained from stereomerically-pureintermediates, reagents, and catalysts by well known asymmetricsynthetic methods.

Geometric isomers can also exist in the compounds of the presentinvention. The symbol

denotes a bond that may be a single, double or triple bond as describedherein. The present invention encompasses the various geometric isomersand mixtures thereof resulting from the arrangement of substituentsaround a carbon-carbon double bond or arrangement of substituents arounda carbocyclic ring. Substituents around a carbon-carbon double bond aredesignated as being in the “Z” or “E” configuration wherein the terms“Z” and “E” are used in accordance with IUPAC standards. Unlessotherwise specified, structures depicting double bonds encompass boththe “E” and “Z” isomers.

Substituents around a carbon-carbon double bond alternatively can bereferred to as “cis” or “trans,” where “cis” represents substituents onthe same side of the double bond and “trans” represents substituents onopposite sides of the double bond. The arrangement of substituentsaround a carbocyclic ring are designated as “cis” or “trans.” The term“cis” represents substituents on the same side of the plane of the ringand the term “trans” represents substituents on opposite sides of theplane of the ring. Mixtures of compounds wherein the substituents aredisposed on both the same and opposite sides of plane of the ring aredesignated “cis/trans.”

The compounds disclosed herein can exist in solvated as well asunsolvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms. In one embodiment, thecompound is amorphous. In one embodiment, the compound is a polymorph.In another embodiment, the compound is in a crystalline form.

The invention also embraces isotopically labeled compounds of theinvention which are identical to those recited herein, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Isotopically labeled compounds of the invention cangenerally be prepared by following procedures analogous to thosedisclosed in the e.g., Examples herein by substituting an isotopicallylabeled reagent for a non-isotopically labeled reagent.

As used in the present disclosure, “NMDA” is defined asN-methyl-d-aspartate.

In the present specification, the term “therapeutically effectiveamount” means the amount of the subject compound that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician. The compounds of the invention are administered intherapeutically effective amounts to treat a disease. Alternatively, atherapeutically effective amount of a compound is the quantity requiredto achieve a desired therapeutic and/or prophylactic effect, such as anamount which results in defined as that amount needed to give maximalenhancement of a behavior (for example, learning), physiologicalresponse (for example, LTP induction), or inhibition of neuropathicpain.

As used herein, “beta-turn motif” or beta-turn refers to a chemicalstructure having C^(α) (alpha carbon) atoms (a carbon atom next to acarbonyl carbon) substantially close, e.g. having a hydrogen bondbetween a donor and acceptor residue, where the donor and acceptorresidue are separated by a distance that corresponds to the distance oftwo or three peptide bonds. A disclosed chemical structure has, forexample, a beta-turn motif when the structure includes bicyclic rings(e.g., bicyclic spiro-lactam) that have restricted rotation, e.g., whichmay be evidenced by weak nOe (nuclear overhauser effect) spectra e.g.,between H3 and H5 atoms.

Compounds

Compounds, e.g. peptide mimetics disclosed herein, in some embodimentsare capable of binding to the NDMA ligand binding core of SEQ ID No. 1.For example, a disclosed peptide mimetic may have e.g. two alpha carbonsthat may be about 6 to about 14 Å, or about 9 to about 14 Å, or about 10to about 13 Å apart. In some embodiments, a contemplated peptide mimeticmay be internally constrained or conformally constrained so that it may,for example, mimic a biologically active conformation of a peptide. Forexample, a disclosed peptide mimetic may include a cyclic amide core,e.g. a bicyclic beta-lactam. For example, a disclosed peptide mimeticmay be a disclosed compound having a two modular units (e.g., a bicyclicbeta-lactam core), wherein each unit can be substituted with a naturallyoccurring amino acid.

For example, a disclosed compound may have a beta-turn motif that isstable upon administration to a patient, e.g. is substantially stablein-vivo or in an aqueous solution. In some embodiments, a disclosedcompound may be capable of forming a hydrogen bond, or may be capable ofbinding to at least one, two, three or four amino acids of SEQ ID NO. 1,e.g. selected from the group consisting of PRO124, THR126, GLU178 andSER180.

A disclosed peptide mimetic may include a small synthetic (i.e.non-peptidyl) conformationally restricted component (e.g. a spirolactammoiety), which may for example, contribute to partial glycine siteagonist activity of the compounds. For example, disclosed compoundsinclude those represented by Formula I:

-   -   and pharmaceutically acceptable salts, stereoisomers, and        N-oxides thereof; wherein T is, independently for each        occurrence, CR₄R₄′, and n is 0, 1, 2 or 3;    -   A is optionally present and is selected from phenyl or pyridine,        wherein A is optionally substituted by one or more substituents        selected from R_(a);    -   R₁ is selected from the group consisting of H, hydroxyl,        —S(O)₂—C₁₋₄alkyl; —SO₂, C₁₋₄alkyl, C₂-C₄alkenyl, phenyl, R₇, or

wherein C₁₋₄alkyl, C₂₋₄alkenyl, or phenyl is optionally substituted byone or more substituents selected from R_(a);

-   -   X is CH or N;    -   R₃ and R₃′ are independently selected from the group consisting        of H, halogen, hydroxyl, phenyl, C₁₋₄alkyl, amido, amine, or        C₂₋₄alkenyl, wherein C₁₋₄alkyl, C₂₋₄alkenyl and phenyl are        optionally substituted by one or more substituents selected from        R_(a);

R₄ and R₄′ are independently selected from the group consisting of H,halogen, hydroxyl, phenyl, C₁₋₄alkyl, amido, amine, C₁₋₄alkoxy orC₂₋₄alkenyl, wherein C₁₋₄alkyl, C₂₋₄alkenyl, C₁₋₄alkoxy, and phenyl areoptionally substituted by one or more substituents selected from R_(a);

-   -   R₂ is selected from the group consisting of H, R₇, —S(O)₂,        S(O)₂—C₁-C₄alkyl, C₁₋₄alkyl, hydroxyl, or phenyl wherein        C₁₋₄alkyl, C₂₋₄alkenyl and phenyl are optionally substituted by        one or more substituents selected from R_(a);    -   R₅ and R₅′ are each independently selected from group consisting        of H, halogen, C₁₋₄-alkyl, C₁₋₄alkoxy, C₂₋₄alkenyl, cyano,        amino, phenyl, and hydroxyl, wherein C₁₋₄alkyl, C₂₋₄alkenyl and        phenyl are optionally substituted by one or more substituents        selected from R_(a);    -   R₇ is selected from group consisting of —C(O)—R₉, —C(O)—O—R₉, or        —C(O)—NR_(d)—R₉,    -   R₉ is selected from the group consisting of H, C₁₋₄alkyl,        phenyl, or heterocyclic, wherein C₁₋₄alkyl, phenyl or        heterocyclic is optionally substituted by 1, 2 or 3 substituents        selected from R_(b)    -   R₈ is selected from group consisting of H, —C(O)—C₁₋₄alkyl or        C(O)—O—C₁₋₄ alkyl, wherein C₁₋₄alkyl is optionally substituted        by 1, 2 or 3 substituents selected from R_(a);    -   R_(a) is selected, independently for each occurrence, from        carboxy, hydroxyl, halogen, amino, phenyl, C₁₋₄alkyl, and        C₁₋₄alkoxy;    -   R_(b) is selected, independently for each occurrence, from the        group consisting of carboxy, hydroxyl, halogen, amino, phenyl,        C₁₋₄alkyl, C₁₋₄alkoxy, and —NH—R_(c);    -   R_(c) is selected, independently for each occurrence from the        group consisting of: —C(O)—O—C₁₋₄alkyl; and —C(O)—C₁₋₄alkyl; and    -   R_(d) is selected, independently for each occurrence, H and        C₁₋₄alkyl;    -   and pharmaceutically acceptable salts, N-oxides or stereoisomers        thereof.

For example, disclosed compounds may include those represented by:

wherein R₁ is C(O)—C₂₋₄alkyl, wherein C₂₋₄alkyl is substituted at onecarbon with NH₂ or —N-carbobenzyloxy and at a different carbon byhydroxyl. For example, R₁ may be C(O)—O—C₁₋₄alkyl (e.g., methyl, ethyl,propyl, wherein C₁₋₄alkyl is substituted by phenyl, and R₃, R₃′, R₅ andR₂ are provided above.

In another embodiment, R₁ and R₂ of formula Ia may each independently beselected from an amino acid, e.g. a L- or D-isomer of an amino acid,e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamicacid, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine and/or valine. For example, R₁ and R₂ may each be independentlyL-Thr or L-Ser, e.g. compounds such as:

wherein R′ is selected from the group consisting of H, or C₁₋₄alkyl.

In an embodiment, R₁ may be carbobenzyloxy, or may be represented by:

wherein X may be N; R₅′ may be H; and R₈ may be —C(O)—C₂₋₄alkyl (e.g.ethyl, propyl, n-butyl, or t-butyl), wherein C₂₋₄alkyl is substituted atone carbon with NH₂ or —N-carbobenzyloxy and at a different carbon byhydroxyl.

In certain embodiments, R₃ may be phenyl (optionally substituted asabove), or may be H. R₂ may be, in some embodiments, a —C(O)—C₂₋₄alkyl,(e.g. ethyl, propyl, n-butyl, or t-butyl), optionally substituted at onecarbon with NH₂ and another carbon with hydroxyl.

For any contemplated R-group that includes C₁₋₄alkyl (e.g. R₁, R₃, R₅),the alkyl may be selected from the group consisting of methyl, ethyl,propyl, n-butyl or t-butyl, and wherein said C₁₋₄alkyl is optionallysubstituted by one, two, or three substituents selected from the groupconsisting of F, Cl, or Br.

Such compounds may have differing isomerizations, and in someembodiments, may be represented by:

and wherein R₁, R₂, R₃, R′₃, and R₅ may be as described above.

In another embodiment, compounds represented by formula II arecontemplated:

and pharmaceutically acceptable salts, stereoisomers and N-oxidesthereof; wherein

-   -   R₁ is selected from the group consisting of H, hydroxyl,        —S(O)₂—C₁₋₄alkyl; —SO₂, C₁₋₄alkyl; R₇, or

-   -   X is CH or N;    -   R₃ and R₃′ are each independently selected from the group        consisting of H, halogen, hydroxyl, phenyl, C₁₋₄alkyl, amido,        amine, or C₂₋₄alkenyl, wherein C₁₋₄alkyl, C₂₋₄alkenyl and phenyl        are optionally substituted by one or more substituents selected        from Ra;    -   R₂ is selected from the group consisting of H, R₇, —S(O)₂,        S(O)₂—C₁₄alkyl, C₁₋₄alkyl, hydroxyl, or phenyl wherein        C₁₋₄alkyl, C₂₋₄alkenyl and phenyl are optionally substituted by        one or more substituents selected from R_(a);    -   R₅ is selected from group consisting of H, halogen, C₁₋₄alkyl,        C₁₋₄alkoxy, C₂₋₄ alkenyl, cyano, amino, phenyl, and hydroxyl,        wherein C₁₋₄alkyl, C₂₋₄alkenyl and phenyl are optionally        substituted by one or more substituents selected from R_(a);    -   R₆ is selected from group consisting of H, halogen, C₁₋₄alkyl,        C₁₋₄alkoxy, C₂₋₄alkenyl, cyano, amino, phenyl, and hydroxyl        wherein C₁₋₄ alkyl, C₂₋₄alenyl and phenyl are optionally        substituted by 1, 2 or 3 substituents selected from R_(a);    -   R₇ is selected from group consisting of —C(O)—R₉, —C(O)—O—R₉, or        —C(O)—NR_(d)—R₉,    -   R₉ is selected from the group consisting of H, C₁₋₄alkyl,        phenyl, or heterocyclic, wherein C₁₋₄alkyl, phenyl or        heterocyclic is optionally substituted by 1, 2 or 3 substituents        selected from R_(b); or    -   or R₁ and R₆, taken together with formula II form:

-   -   R₈ is selected from group consisting of H, —C(O)—C₁₋₄alkyl or        C(O)—O—C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted by        1, 2 or 3 substituents selected from R_(a);    -   R_(a) is selected, independently for each occurrence, from        carboxy, hydroxyl, halogen, amino, phenyl, C₁₋₄alkyl, and        C₁₋₄alkoxy;    -   R_(b) is selected, independently for each occurrence, from the        group consisting of carboxy, hydroxyl, halogen, amino, phenyl,        C₁₋₄alkyl, C₁₋₄alkoxy, and —NH—R_(c);    -   R_(c) is selected, independently for each occurrence,        —C(O)—O—C₁₋₄alkyl; and —C(O)—C₁₋₄alkyl; and    -   R_(d) is H or C₁₋₄alkyl.

In an exemplary embodiment, the R₁ moiety of Formula I, II, Ia or Ib maybe selected from the group consisting of:

Exemplary compounds include

Disclosed herein are compounds selected from the group consisting of:

(where n is 0, 1, 2 or 3), and

or pharmaceutically acceptable salts thereof.and pharmaceutically acceptable salts, stereoisomers, or N-oxidesthereof.

The compounds of the present disclosure and formulations thereof areintended to include both a D-isomeric form, an L-isomeric form, or aracemic mixture (both D- and L-isomeric forms) of any one or more of thecompounds. In addition, the formulations of the compounds are intendedto include any combination or ratio of L-isomeric forms to D-isomericforms of one or more of the analogs described herein. These and otherformulations of the disclosed compounds comprising a greater ratio ofthe D- and/or L-isomeric analog form may posses enhanced therapeuticcharacteristic relative to racemic formulations of a disclosed compoundsor mixture of compounds. For example, disclosed compounds may beenantiomers, e.g.:

Disclosed compounds may provide for efficient cation channel opening atthe NMDA receptor, e.g. may bind or associate with the glutamate site ofthe NMDA receptor to assist in opening the cation channel. The disclosedcompounds may be used to regulate (turn on or turn off) the NMDAreceptor through action as an agonist.

Other compounds contemplated herein include those having a cyclic amidecore. Other exemplary compounds may be, in an embodiment, peptides, orin another embodiment, peptide mimetics. Contemplated compounds includethose represented by:

wherein,R₁ is H or benzyl group;R₄ is H or benzyl group;R₅ is

R₆ is

R₂ is H or CH₃;R₃ is H or CH₃; and stereoisomers, or pharmaceutically acceptable saltsor N-oxides thereof. Exemplary compounds include (SEQ ID NOS 2-3,respectively in order of appearance):

The compounds as described herein may be glycine site NMDA receptorpartial agonists. A partial agonist as used in this context will beunderstood to mean that at a low concentration, the analog acts as anagonist and at a high concentration, the analog acts as an antagonist.Glycine binding is not inhibited by glutamate or by competitiveinhibitors of glutamate, and also does not bind at the same site asglutamate on the NMDA receptor. A second and separate binding site forglycine exists at the NMDA receptor. The ligand-gated ion channel of theNMDA receptor is, thus, under the control of at least these two distinctallosteric sites. Disclosed compounds may be capable of binding orassociating with the glycine binding site of the NMDA receptor. In someembodiments, disclosed compounds may possess a potency that is 10-foldor greater than the activity of existing NMDA receptor glycine sitepartial agonists. For example, disclosed compounds may possess a 10-foldto 20-fold enhanced potency compared to GLYX-13. GLYX-13 is representedby (SEQ ID NO: 5):

For example, provided herein are compounds that may be at least about20-fold more potent as compared to GLYX-13, as measured by burstactivated NMDA receptor-gated single neuron conductance (I_(NMDA)) in aculture of hippocampal CA1 pyramidal neurons at a concentration of 50nM. In another embodiment, a provided compound may be capable ofgenerating an enhanced single shock evoked NMDA receptor-gated singleneuron conductance (I_(NMDA)) in hippocampal CA1 pyramidal neurons atconcentrations of 100 nM to 1 μM. Disclosed compounds may have enhancedpotency as compared to GLYX-13 as measured by magnitude of long termpotentiation (LTP) at Schaffer collateral-CA-1 synapses in in vitrohippocampal slices.

Preparation of Compounds

In some embodiments, a disclosed compound, e.g. a peptide mimetic havinga beta-turn motif capable of binding to the NMDA ligand binding core ofSEQ ID NO 1, may be formed by incorporating one or more dehydro aminoacids within a peptide. For example, peptides containingdehydrophenylalanine and/or dehydroleucine and/or alpha-aminobisobutyricacid may be incorporated to make a peptide mimetic having a beta-turnmotif.

In another embodiment, a disclosed compound may be formed using aconstrained bicyclic beta-turn dipeptide (BTD) motifs. This approach isbased on the replacement of dipeptide component by incorporation of acarboxyl and an amino group in geometrically suitable positions forpeptide coupling (see figure below) for inducing turns.

For example, a dipeptide component such as one or more azabicyclononanesmay be incorporated to produce a structure having a beta-turn motif. Inanother example, a disclosed peptide mimetic may include a corestructure as exemplified below, e.g. in place of two, three, or fouramino acids:

In some embodiments, an azacycloalkeane may mimic a beta turn mimetic,e.g.:

The following schemes are representative synthetic routes that may beused to prepare disclosed compounds and intermediates thereof.

Ceric ammonium nitrate, or “CAN”, is the chemical compound with theformula (NH₄)₂Ce(NO₃)₆. This orange-red, water-soluble salt is widelyused as an oxidizing agent in organic synthesis. This compound is usedas a standard oxidant in quantitative analysis.

PMP refers to p-methoxybenzylidene; Cbz refers to a carbobenzyloxyradical that can be depicted as:

Compositions

In other aspects, formulations and compositions comprising the disclosedcompounds and optionally a pharmaceutically acceptable excipient areprovided. In some embodiments, a contemplated formulation comprises aracemic mixture of one or more of the disclosed compounds.

Contemplated formulations may be prepared in any of a variety of formsfor use. By way of example, and not limitation, the compounds may beprepared in a formulation suitable for oral administration, subcutaneousinjection, or other methods for administering an active agent to ananimal known in the pharmaceutical arts.

Amounts of a disclosed compound as described herein in a formulation mayvary according to factors such as the disease state, age, sex, andweight of the individual. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for themammalian subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier.

The specification for the dosage unit forms of the invention aredictated by and directly dependent on (a) the unique characteristics ofthe compound selected and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin.

The compounds can be administered in a time release formulation, forexample in a composition which includes a slow release polymer. Thecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations aregenerally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating thecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In accordance with an alternative aspect of the invention, a compoundmay be formulated with one or more additional compounds that enhance thesolubility of the compound.

Methods

Methods for treating cognitive disorders and for enhancing learning isprovided. Such methods include administering a pharmaceuticallyacceptable formulation of one or more of the disclosed compounds to apatient in need thereof. Also contemplated are methods of treatingpatients suffering from, memory deficits associated with aging,schizophrenia, special learning disorders, seizures, post-strokeconvulsions, brain ischemia, hypoglycemia, cardiac arrest, epilepsy,migraine, as well as Huntington's, Parkinson's and Alzheimer's disease.

Other methods contemplated include the treatment of cerebral ischemia,stroke, brain trauma, brain tumors, acute neuropathic pain, chronicneuropathic pain, sleep disorders, drug addiction, depression, certainvision disorders, ethanol withdrawal, anxiety, and memory and learningdisabilities. In yet another aspect, a method for enhancing pain reliefand for providing analgesia to an animal is provided

EXAMPLES

The following examples are provided for illustrative purposes only, andare not intended to limit the scope of the disclosure.

Example 1 Synthesis of Pyrrolidine-Derived Spiro β-Lactam Derivatives

The following reaction sequence was used (Scheme A) to synthesize SpiroLactams. Hexahydro-1,3,5-triazines, Cbz-L-proline acid chloride andN-(Cbz) O-(benzylether)-L-threonine acid chloride as starting materials.

TABLE 1 HPLC Mass Lot # Structure Quantity % Purity (M⁺ H) HNMR 4

20 mg 93 261 YES 5(AK-51)

150 mg  — 127 YES (>95% purity) 8

17 mg 73 496 —

Example 2 Synthesis of Compounds and Intermediates

Spiro Lactam 3. The synthesis of C4 unsubstituted spirolactam 3 wasconducted via Staudinger reaction of methyleneimine derived fromtriazine 2. The [2+2]-cycle addition reaction between the ketene derivedfrom Cbz-L-proline acid chloride and the methyleneimine was carried outin the following way: ketene was generated by dehydrochlorination of theacid chloride with triethylamine at −40° C. for 45 min, and then adichloromethane solution of triazine 2 and boron trifluoride etherate(which depolymerize the triazine) was added. After 12 hours, thecorresponding spirolactam 3 was obtained as a mixture of enantiomers,with 30 to 50% yield. The oxidative removal of the PMP group from spirolactam 3 in the presence of CAN gave the N-unsubstituted derivativespirolactam 4, which upon treatment with Pd(OH)₂/C gave thecorresponding spirolactam intermediates 5.

Spiro lactam 4 was obtained in 93% purity (HPLC) after purification bychromatography on silica gel. Spiro lactam 5 were obtained withpurities >90% purity (by NMR) after chromatography on silica gel usinggradient elution 20% to 70% Ethyl Acetate Cyclohexane, in 50% yield.

Example 3 Synthetic Routes to Intermediate Compounds

Triazine 2. To a solution of p-anisidine (24.6 g, 200 mmol.) in amixture (500 mL) of ethyl acetate/water (1:1), cooled at 0° C., anaqueous solution (17 mL) of formaldehyde (37%) was added. The reactionmixture was stirred for 3 hours at 0° C. then 1 hour at roomtemperature, and the organic layer was separated, washed with water (50mL), and dried over Na₂SO₄. The solvent was removed under vacuum, and awhite solid was obtained. This solid was washed once with diethyl etherto provide 26.3 g (solid was dried at 40° C. overnight) of pure triazine2 in 97% yield.

Spiro lactam Intermediates 3. To a stirred solution of theN-benzyloxycarbonyl L-proline acid chloride (5 g, 18.7 mmol.) in drydichloromethane (65 mL) cooled to −40° C., was added dropwise drytriethylamine (10.4 mL, 74.7 mmol.). The solution became yellow toconfirm that the ketene was formed.

After 45 min at −40° C., a purple solution of triazine 2 (2.52 g, 6.16mmol.) and BF₃ OEt₂ (2.37 mL, 18.7 mmol.), previously mixed in CH₂Cl₂(35 mL), was added dropwise. The mixture was allowed to warm slowly toroom temperature overnight and then quenched with saturated aqueousNaHCO₃. The aqueous layer was extracted twice with CH₂Cl₂ (20 mL); thecombined organic layers were washed with brine (20 mL) and dried overanhydrous Na₂SO₄. The solution was then concentrated and purified bycolumn chromatography over silica gel using gradient elution100%/cyclohexane to 20% ethyl acetate/cyclohexane to give 7.01 g of pureproduct with 37% yield.

Spiro lactam Intermediates 4. To a stirred solution of spirolactam 3(2.4 g, 6.55 mmol.) in acetonitrile (49 mL) at −10° C., was addeddropwise over 1 hour CAN (10.8 g, 19.6 mmol.), previously dissolved inH₂O (30 mL). After the addition was complete, the mixture was stirredfor 45 min (TLC showed no remaining starting material). The reactionmixture was diluted with ethyl acetate (100 mL) and saturated NaHCO₃ (50mL). To the organic layer was added water (100 mL) and solid sodiumbisulfite (20 eq). The organic layer was washed with brine and driedover anhydrous Na₂SO₄. The solution was then concentrated and purifiedby column chromatography over silica gel using gradient elution100%/cyclohexane to 50% ethyl acetate/cyclohexane to give 0.87 g of pureproduct in 50% yield.

Spiro lactam Intermediates 5 (AK-51). 0.5 g of 4 were dissolved in 20 mLof ethyl acetate and transferred via cannula to a flask under H2 (1 atm)containing 50 mg of 10% Pd(OH)₂—C catalyst. The mixture was stirred forovernight under H₂ at 50 PSi and then the catalyst was filtered offthrough celite. The organic layer was concentrated and purified bychromatography on silica gel to afford 120 mg of product in 50% yield.

N-(Cbz)-O-(benzyl ether)-L-threonine acid chloride 7. To a stirredsolution of N-(Cbz)-O-(benzyl ether)-L-threonine (0.95 g, 2.7 mmol.) indry ether (27 mL) was added PCl5 (0.61 g, 2.9 mmol.) and the mixture wasstirred for 3 hours at room temperature. Then the solvent was removedwith high vacuum at room temperature. Toluene was added and removed asabove. The crude white solid was used without any purification for thecoupling reaction.

Spiro lactams Intermediates 8 and 9. To a stirred solution ofspirolactam 4 (200 mg, 0.76 mmol.) in dry THF (4 mL) at −78° C. wasadded BuLi (0.32 mL, 0.80 mmol. in hexane) dropwise. After addition wascomplete, the mixture was stirred at −78° C. for 1 hour.N-(Cbz)-O-(benzyl ether)-L-threonine acid chloride 7 in THF (4 mL) wasadded at −78° C. The mixture was stirred for overnight from −78° C. toroom temperature.

The reaction mixture was quenched with saturated NH₄Cl (10 mL) and ethylacetate (10 mL) was added. The water layer was extracted twice withethyl acetate. The combined organic layers were dried with MgSO₄ andconcentrated to give 0.44 g of crude product. The crude product waseluted through silica gel with a gradient from 100% CH₂Cl₂ to 2%MeOH/CH₂Cl₂ giving fractions that ranged in purity from 44% to 73%. Thisreaction was repeated on 0.28 g of spirolactam 4 and gave afterchromatography fractions with purities that ranged from 50% to 73%.

Example 4 NMDA Receptor Binding Assay

Tissue Preparation:

Crude synaptic membranes were prepared from rat hippocampi or from ratforebrains (male Sprague-Dawley rats) and washed extensively to removeendogenous amino acids, as previously described by Ransom and Stec(1988). Briefly, the crude synaptic membranes were resuspended in 20volumes of 5 mM Tris-HCl buffer, pH 7.4 (for use in [³H]TCP-bindingexperiments), or in 20 volumes of 5 mM Tris-acetate buffer, pH 7.4 (foruse in [³H]glycine-binding studies) and homogenized using a Polytron(Virtis shear; Virtis, N.Y., U.S.A.). Membranes were then pelleted bycentrifugation at 48,000 g for 20 min. This step was repeated twice andthe homogenate was stored at −70° C. in the same buffer. Before eachuse, homogenates were thawed at room temperature, pelleted, and washedfour additional times. For the [³H]glycine experiment, the pellet wasfirst incubated for 30 min at 25° C. in 5 mM Tris-acetate buffercontaining 0.04% Triton X-100 and then washed four times byhomogenization and centrifugation. The final washed membranes wereresuspended at concentrations of 2-3 mg/ml in either 5 mM Tris-HClbuffer or 5 mM Tris-acetate buffer.

TCP binding assays: Measurements of specific [³H]TCP binding wereperformed as described previously (Haring et al., 1986, 1987; Kloog etal., 1988a). Final reaction mixtures consisted of 50-100 μg of membraneprotein in 200 μl of 5 mM Tris-HCl buffer and contained either [³H]TCP,or [³H]TCP and the appropriate concentration of NMDA-receptor ligands ormAbs. Reactions were initiated by the addition of the membranes to thereaction mixtures. Unless otherwise indicated, binding assays wereperformed under nonequilibrium conditions at 25° C. for 1 h. Nonspecificbinding was determined in parallel samples containing 100 μM unlabeledPCP. Binding reactions were determined by filtration on Whatman GF/Bglass filters that had been pretreated with 0.1% polyethyleneimine for 1h.

The dissociation of [³H]TCP from its membrane-binding site was measuredafter equilibrating the receptors with 20 nM [³H]TCP for 120 min. Thedissociation reaction was initiated by the addition of 100 μM unlabeledPCP in the presence and absence of NMDA-receptor ligands or mAb.Reactions were terminated immediately (zero time) and after incubationfor the additional periods of time indicated.

The effects of the three compounds were examined on 1) NMDAreceptor-gated single neuron conductance (I_(NMDA)) in hippocampal CAIpyramidal neurons and 2) the magnitude of long-term potentiation (LTP)and long term depression (LTD) at Schaffer collateral CA1 synapses, inin vitro hippocampal slices. GLYX-13 has been reported to exibit a lowconcentration (1-10 μM) enhancement of burst-activated I_(NMDA) and LTP,while simultaneously reducing LTD and single pulse evoked I_(NMDA). Ahundred fold higher GLYX-13 concentration of 100 μM converted toreducing LTP and burst I_(NMDA), and no longer affected LTD.

Compound B showed a 20-fold enhancement in potency compared to GLYX-13.50 nM of this compound markedly enhanced both single shock (1A) andburst evoked (1B) I_(NMDA), as well as doubling the magnitude of LTP(1E). In contrast, 1 μM NRX-10,050 significantly reduced both singleshock (1C) and burst evoked ((ID) I_(NMDA), reminiscent of 100 μMGLYX-13. (See FIG. 2).

AK-51 exhibited less potency than compound B, but a wider concentrationrange in its stimulatory actions (FIG. 3). Both 100 nM (2A) and 1 μMNRX-10,051 enhanced single-shock evoked I_(NMDA), while 1 uM NRX-10,051doubled the magnitude of LTP (2D), while not altering LTD (2E).

AK-52 produced only a mild enhancement of single-shock evoked I_(NMDA)at a low concentration (100 nM; 3A), which converted to significantreduction in I_(NMDA) at a 1 uM concentration (3B). 100 nM AK-52produced an enhancement of LTP similar in magnitude to compound B andAK-51, but this converted to a slight, but significant, reduction in LTPat the 1 μM concentration, without altering LTD.

These three compound showed about a 20-fold enhancement in potencycompared to GLYX-13. Compound B is the most potent enhancer of I_(NMDA)at low concentrations (50 nM). While AK-51 enhancement of I_(NMDA) wassmaller in magnitude, this effect remained when the AK-51 was increased10-fold (100 nM to 1 μM). The AK-52 was the weakest enhancer ofI_(NMDA), and this effect reversed more quickly to a frank reduction inI_(NMDA).

These compounds enhanced the magnitude of LTP to similar extents,approximately to a doubling. GLYX-13 was the only compound that couldsimultaneously increase LTP and reduce LTD: AK-52 did not affect LTD,even at a concentration that reduced I_(NMDA). GLYX-13 can selectivelyenhance I_(NMDA) mediated by NMDA receptors containing NR2A/B subunits,and these receptors are localized to extrasynaptic loci and are morestrongly activated by neuronal bursts that induce LTP. While all of thetested compounds have potent effects on LTP and I_(NMDA), the lessereffects on LTD suggest that they have increased selectivity for NR2A/Bcontaining NMDA receptor glycine sites than the GLYX-13.

Example 5 T-Maze Learning Model

Male 3 month old Fisher 344 X Brown Norway F1 cross rats (FBNF1) wereused for this study. The t-maze was constructed with arms (45 cm long×10cm wide×10 cm high) made of black Plexiglas enclosing the maze. Twoplastic bottle caps, lined with wire mesh, were secured to the end ofeach goal arm in which the food reward (Cheerios, 100 mg/piece) wasplaced. Before the start of training, animals were gradually deprived offood to approximately 85% of their free feeding weight. On threesuccessive days before the start of training, animals were habituated tothe t-maze with food located throughout the maze. On the first day oftraining, animals were rewarded for right arm choices and were trainedto a criterion of 9 out of 10 consecutive correct choices. On the secondday of training, animals were rewarded for left arm choices, and weretrained to a criterion of 9 out of 10 consecutive correct choices. Onthe subsequent testing day, animals were given injections of AK51 (0.3,1, 3, 10, 30 mg/kg p.o.), or DMSO vehicle (1 mg/ml; Sigma, Saint LouisMo.) in a blind manner via gastric gavage (4″, 16-ga; BraintreeScientific, Braintree Mass) 60 min prior to the start of testing (n=8-9per group. On the first trial of testing, both arms were baited withfood and for the subsequent 20 trials only alternating choices (oppositeof the animal's previous choice) were rewarded (˜30 sec inter-trialinterval). The number of trials to criterion (5 consecutive correctchoices) was calculated for each animal. Data was analyzed by ANOVAfollowed by Fisher PLSD post hoc tests comparing individual drug dosesto vehicle (α=0.05).

FIG. 5 depicts mean (±SEM) trials to criterion in the alternating T-mazetask (20 trials) in food deprived 3 month old rats. Animals wereinjected p.o. with 0, 0.3, 1, 3, 10, or mg/kg AK051 in DMSO vehicle(n=8-9 per group) 60 min before the start of testing. ***P<0.001,**P<0.01, Fisher PLSD post hoc vs. vehicle

Example 6 Formalin Test of Neuropathic Pain

Experiments were conducted as previously described (Abbott et al. Pain,60, 91-102, 1995; Wood et al., Neuroreport, 19, 1059-1061 2008). Male 3month old Fisher 344 X Brown Norway F1 cross rats (FBNF1) were used forthis study. Before the start of testing, animals were habituated to thetesting chamber (30×30×60 cm opaque plexiglass) for 10 min each day over2 consecutive days. On the testing day, animals were given injections ofAK51 (0.3, 1, 3, 10, 30 mg/kg p.o.), or DMSO vehicle (1 mg/ml; Sigma,Saint Louis Mo.) in a blind manner via gastric gavage (4″, 16-ga;Braintree Scientific, Braintree Mass.) 60 min prior to formalininjections (n=8-9 per group). Animals were placed into the testingchamber 10 min prior to formalin injection. For the formalin injection,rats were manually restrained and given a subcutaneous injection of 1.5%formalin (50 μL with a 26-ga needle; Sigma, Saint Louis Mo.) into thelateral footpad on the plantar surface of the left hind paw. Afterformalin injections rats were placed back into the testing chambers.Animals were videotaped from below with the aid of an angled mirror for50 min post formalin injection. Total time spent licking the injectedpaw and total number of injected paw flinches during the late phase(30-50 min post formalin injection) were quantified off-line in a blindmanner by a trained experimenter with high (r>0.9) inter- andintra-rater reliability for both measures. All animals were euthanizedby CO₂ immediately after testing. Data was analyzed by ANOVA followed byFisher PLSD post hoc tests comparing individual drug doses to vehicle(α=0.05). FIG. 6 depicts mean (±SEM) % Analgesia defined as % reductionin flinches in the late phase response (30-50 min) after intraplantarformalin injection (50 μL of 1.5% formalin).

Example 7 Oral Formulations Enhancing Learning and Memory

An oral preparation of AK-51, was prepared in dimethylsulfoxide (DMSO).All doses were administered in a volume of 300 μl. The animals were thenfed p.o. by gavage (force fed by mouth with an inserted feeding needle)a volume calculated to deliver to the animal a defined dose based onbody weight as follows 0.0 mg/kg 300 μL DMSO (vehicle); 0.3 mg/kg, 300μL in DMSO; 1.0 mg/kg, 300 μL in DMSO; 3.0 mg/kg, 300 μL in DMSO; 10.0mg/kg, 300 μL in DMSO; 30.0 mg/kg, 300 μL in DMSO.

Animals were injected 60 minutes before the start of testing with one ofthe dose amounts recited above. Then, an alternating T-maze task (20trials) was used to asses learning behavior in the animals. Thisprotocol is described at Example 5. Briefly, the T-maze is a choicetask. The subject rat was placed in the base of the “T”. Following ashort delay, it was allowed to explore the maze and choose to entereither the right or left arms. The choice is scored according to varietyof criterion, including spontaneous alternation, cued reward, or toindicate a preference. Based on the criterion used in this study, theT-maze was used to test learning and memory. Food placed at one end ofthe maze was used as the positive reinforcer for each animal test.

Animals given a 1.0 mg/kg dose by mouth of AK-51 demonstrated astatistically significant enhancement of learning behavior in the T-mazetest (P<0.001). Animals given a 3.0 mg/kg dose by mouth of thenon-peptide analog NRX-10,051 also demonstrated a statisticallysignificant enhancement of learning behavior in the T-maze test(P<0.01).

Example 8 Isomers

The two different isomers of AK-55 was used in a NDMA binding assay asin Example 4. One isomer of AK-55 potently enhances NMDA while the otherdoes not. FIG. 7A indicates the time course of effect of 15 min bathapplication of 1 μM AK55 (solid bar) on normalizedpharmacologically-isolated NMDA receptor-gated current in CA1 pyramidalneurons under whole-cell recording (mean±SEM, n=6). B: Time course ofeffect of 15 min bath application of 1 μM AK55 (solid bar) on normalizedpharmacologically-isolated NMDA receptor-gated current in CA1 pyramidalneurons under whole-cell recording (mean±SEM, n=7). C: Time course ofeffect of bath application of 1 μM AK6 (solid bar, filled circles, n=8)compared to untreated control slices (open circles, n=8) on themagnitude of long-term potentiation (LTP) of extracellular excitatorypostsynaptic potential slope (mean±SEM fEPSP) induced by high-frequencySchaffer collateral stimulation (2×100 Hz/500 msec).

Example 9 Biochemical Assays

Table B depicts the results of binding assays against various targetswith AK51:

TABLE B Target Species Concentration % Inhibition Glutamate, AMPA rat 10μM −8 Glutamate, Kainate rat 10 μM −13 Glutamate, Metabotropic, mGlu_(s)human 10 μM −7 Glutamate, NMDA, Agonism rat 10 μM 27 Glutamate, NMDA,Glycine rat 10 μM −6 Glutamate, NMDA, rat 10 μM −5 PhencyclidineGlutamate, NMDA, Polyamine rat 10 μM −14 Glutamate, Non-Selective rat 10μM −10 Glycine, Strychnine-Sensitive rat 10 μM 4 Potassium Channel hERGhuman 10 μM 3

Example 10 Identification of β Turn in Spiro Compounds

Proton 1-D experiments ¹H, ¹³C, DEPT, homo nuclear 2-D experiments(DQF-COSY, TOCSY, NOESY) and Hetero nuclear experiments HSQC and HMBC inDMSO-D₆ at 30 degree Celsius are conducted to confirm the exact carbonchemical shifts and protons chemical shifts of the spiro compound:

Chemical shifts are observed as follows: ¹H, DMSO-d₆, 600 MHz, δ in ppm,TMS at 0.00 ppm: 8.72 (bs, 1H), 3.47dd, 2H), 3.37 (t, 2H), 2.21 (m, 2H),2.02 (m, 1H), 1.89 (m, 1H) (see FIG. 12);

¹³C, DMSO-d₆, 150 MHz, δ in ppm, reference DMSO at 39.5 ppm: 169.6,68.7, 45.6, 40.7, 32.9, 22.4.

The chemical shift of the amide proton was located at 8.72 ppm as abroad singlet, and cross peaks were observed between 8.72 and 3.37. Thisfinding established the chemical shift of H-3 at 3.47 ppm. Weak nOebetween 3.37 and 2.21 ppm indicates the populations of H-5 at 2.21 ppm.Total correlation was found from 3.37 to 2.21 ppm through 2.02 1nd 1.89ppm; nOe correlation was observed between 2.21, 2.02, 1.89 and 3.37 ppm.This finding was understandable as the resonances H-6 and H-7 are: H-6(2.02 and 1.89 ppm), H-7 (3.37 ppm). The heteronuclear 2-D experiments(HSQC and HMBC) also confirmed the chemical shifts of protons andcarbon.

The chemical shift of individual protons and carbon were observed asfollows:

8.72 (bs, H-2, 1H), 3.47 (dd, H-3, 2H), 3.37 (t, H-7, 2H), 2.21 (m, H-5,2H), 2.02 (m, H-6, 1H), 1.89 (m, H-6′1H)

169.6 (C-1), 68.7 (C-4), 45.6 (C-7), 40.7 (C-3), 32.9 (C-5), 22.4 (C-6)

A weak nOe between H-3 and H-5 is suggestive of restricted rotation ofthe rings with respect to each other. The absence of hydrogen bonds inwhich the donor and acceptor residues i (i±3) and also absence of longrange nOe (C^(α) atoms<7A°) between two rings indicates there is nosignificant secondary fold.

Example 11

Proton 1-D experiments ¹H, ¹³C, DEPT, homo nuclear 2-D experiments(DQF-COSY, TOCSY, NOESY) and Hetero nuclear experiments HSQC and HMBC inDMSO-D₆ at 30 degree Celsius are conducted to confirm the exact carbonchemical shifts and protons chemical shifts of the spiro compound

The ¹H NMR in DMSO is shown in FIG. 13. A N15-HSQC experiment at 600 MHzcan be performed to confirm amide chemical shifts.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference, including thefollowing incorporated by reference:

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What is claimed is:
 1. A compound represented by:

wherein R⁴ is H; R¹ is benzyl; R⁵ is

R⁶ is

R² is H or CH₃, R³ is H or CH₃, and stereoisomers, N-oxides orpharmaceutically acceptable salts thereof.
 2. A pharmaceuticallyacceptable composition comprising a compound of claim 1, and apharmaceutically acceptable excipient.
 3. The compound of claim 1,wherein R² and R³ are CH₃.
 4. The compound of claim 1, wherein R² is CH₃and R³ is H.
 5. The compound of claim 1, represented by:


6. A compound represented by: